The history of macromolecular crystal growth extends more than 150 years (a review from McPherson, 1991). Crystallization of hemoglobin from earthworm's was first observed Hiinefeld in 1840 by pressing two slides of glass and allowed to dry very slowly. This revealed that protein crystals could be obtained by the controlled evaporation of a concentrated protein solution. Funke was the first person devised a successful and reproducible method for the growth of hemoglobin crystals in 1851. He described in-vitro crystallization of hemoglobin from human and animals. However, urease enzyme was crystallized by Summer, which the enzyme was exposed into 30% of acetone at cold temperature.
Crystallization of macromolecules is a complex process based on finding individual conditions and parameters leading to formation of crystal. Crystallization is one of several means by which has thermodynamic driving force that pushes the system (supersaturated solution) back to its quilibrium point reduction of salute concentration. The general processes by which substances crystallize are similar for molecules of both microscopic (salts and small molecules) and macroscopic (proteins, DNA, RNA) dimensions.
There are three stages of crystallization common to all systems such as nucleation, growth and cessation of growth. Nucleation is a process by which molecules or noncrystalline aggregates are free in solution come together to produce a thermodynamic stable aggregate with a repeating pattern. Basically in nucleation the molecules must overcome an energy barrier to form a periodically ordered aggregate or critical size.
Crystal growth generally starts at solute concentrations sufficient for nucleation to occur, and continues at concentrations beneath the nucleation threshold. The growth of crystals from nuclei is also strongly influenced by diffusion and convection effects. As with nucleation, increased protein concentration results in increased growth rates. However, in the metastable region the previously foamed nuclei will continue slowly and orderly to produce the fewest and largest single crystal. At eventually, depletion of nutrient is observed from surroundings of the single crystal.
Cessation of growth of crystals can occur for a multitude of reasons. If there should be a decrease in concentration of crystallizing solute to the point where the solid and solution phases reach exchange equilibrium. The addition of more solute can result in continued crystal growth. However, when some crystals reach a certain size beyond which growth does not proceed irrespective of salute concentration.
The complexity of crystallization problems are well presented in a schematic phase diagram of protein crystallization from McPherson, 1999 [1]
Protein crystallization is necessary for structure elucidation by X-ray diffraction. The crystallization of protein can be divided into two stages: 1) initial screening to obtain any kind of crystals or promising precipitates, and 2) optimization to improve the crystals. The appearance of crystals, even microcrystals of the smallest size or poorest quality, represents the single most important point in achieving the ultimate objective, the determination of a macromolecular structure of X-ray diffraction analysis. Thus, in the process of developing screening condition, it is desirable to create degree of supersaturation where nuclei are likely to form with reasonable concentration of protein but just below the concentration which produces uncontrollable precipitation. A sample scheme for finding optimum crystallization condition is to determine the effect of pH on precipitation with a given precipitant at various temperatures and different precipitating agents.
The ultimate goal of protein expression and purification was a single crystal that diffracts well towards structural determination through X-ray diffraction analysis. The high amount of protein with high purity (˜99%) is the crucial step prior to protein crystallization. Protein expression systems available in the market simplify the basic necessity in providing sufficient yield of target protein heterologously in prokaryotic, eukaryotic or mammalian systems. As a consequence, low protein yield problem from the wild-type bacteria, particularly heat-stable lipase from Geobacillus spp. could be solved by manipulating gene expression. Besides, protein purification strategies are important in supplying sufficient target protein with high purity. Generally, the purification step is simplified if the targeted protein expressed as fusion protein.
Although a few lipase crystal structures of Pseudomonas spp., Chromobacterium viscosum ATCC 6918 and Bacillus spp. were reported so far, but none of them were derived from crystals through high temperature crystallization. It is therefore an object of the present invention, to provide a quality growth of crystal, whereby the said growth is affected by precipitants, pH, protein concentration and at high temperature.
According to the present invention, this object is solved by providing a proper selection of crystal growth conditions by precipitants, pH, protein concentration and at high temperature, wherein the affect of high temperature is possible for the growth of thermostable T1 lipase crystals.